The present invention relates to a nickel-based reagent that covalently binds to a specific nucleotide. The present invention also relates to a nickel-based salicylimine complexed to a histidine containing organic compound, as well as a nickel-based complex comprising a detectable label.
There are a variety of methods for detecting DNA/RNA-protein interactions and some depend on cross-linking. However, to the inventors' knowledge, there is no current technology that uses a nickel-based complex to covalently bind to a specific nucleotide and detect such binding and optionally isolating the DNA regions through use of a labeled complex.
A salicylimine containing nickel complex or any square planar, 4-coordinate nickel complex system linked with any detectable label is sufficient to practice the present invention. Moreover, the salicylimine containing nickel group complexed with the N-terminus of a polypeptide where the terminal amino acid is followed by a histidine or histidinyl residue results in a nickel based reagent that has additional desirable functional capabilities. If this terminal amino acid sequence is not naturally derived, then genetic manipulation may be used to append such a terminus on to any polypeptide of interest. For non-natural amino acids, chemical synthesis is preferred. Further, a semi-synthesis approach can be taken. Combining genetic and chemical means to obtain an amino acid sequence complexed with the salicylimine containing nickel complex is encompassed by the present invention. In addition, this complex may be used to add an aromatic aldehyde selectively to a nucleotide sequence which can be used as a handle for further modification or identification. Furthermore, whereas single-stranded regions of nucleic acids typically react more readily with various chemical and biochemical reagents, their detection and characterization method require prior knowledge of the target sequence. A nickel complex-identifier (such as biotin) of the invention will allow for purification and identification of the nucleic acid sequence without prior knowledge of the reactive sequence. Whereas there is no current reagent that selectively couples to and stays attached to the bound nucleotide, the nickel reagent of the invention allows for the detection of the bound sequence as well as isolation of the nucleotide region.
Shearer, J. M. and Rokita, S. E., Bioorg. Med. Chem. Lett. 9, 501-504, (1999), discloses the preparation of diamine compound for synthesis of a water-soluble Ni (II)-salen (salicylaldehyde) complex. This reference discloses a synthesis method for a simple, unconjugated Ni (Salen) called TMAPES. However, this reference does not disclose the attachment of a label or an adduct to this complex so that the complex can be detected biochemically.
Liang et al., J. Am. Chem. Soc., 120, 248-257 (1998), discloses a Ni (II) linked metallopeptide that is used to selectively degrade DNA through a minor groove binding interaction. However, this reference does not disclose or suggest the covalent linkage between a nickel containing complex and a specific nucleotide.
Routier et al., Bioconjugate Chem., 8, 789-792 (1997), discloses a conjugate between a salen and any biologically active molecule. The complex uses copper. However, such a copper conjugate cleaves nucleic acids non-specifically while non-covalently binding to the backbone of the nucleic acid. This is contrary to the present invention, which is directed to specific coupling of the nickel-containing complex to a nucleotide base.
Routier et al., Biooragnic & Medicinal Chemistry Letters, Vol. 7, No. 1, pp. 63-66 (1997), discloses a Ni-Salen complex. However, in this reference, coupling the Ni-Salen complex with the terminal N group would typically create a neutral compound that would be insoluble and not useful in coupling to nucleic acid bases.
Bhattacharya and Mandal, J. Chem. Soc., Chem. Common., 1995, pp. 2489-2490, discloses a Co-Salen chemistry. Again, this reference does not disclose formation of an adduct with nucleic acid.
Tanaka et al., Bull. Chem. Soc., Jpn. 70, 615-629 (1997), discloses the synthesis of a Cu-Salen molecule. This reference does not disclose or suggest formation of an adduct with nucleic acids.
Gravert and Griffin, J. Org. Chem., 58, 820-822 (1993), discloses a Mn-Salen complex. This reference does not disclose the formation of an adduct with nucleic acids.
Chen and Lu, Journal of the Chinese Chemical Society, 45, 611-617 (1998), discloses a Ru-Salen complex. This reference does not disclose or suggest the formation of an adduct with nucleic acids.
Jacquet et al., J. Am. Chem. Soc. 119, 11763-11768, (1997), discloses a Ru metal-Salen complex which requires photo-activation.
Gravert and Griffin, Inorg. Chem., 35, 4837-4847 (1996), discloses a Mn-Salen complex. However, this reference does not disclose the formation of an adduct with nucleic acids.
Brown et al., Biochemistry, 34, 4733-4739 (1995), discloses a nickel-His-tag and a nickel-glycylglycylhistidine for protein-protein cross-linking. However, the reference does not disclose nickel-dependent protein-nucleic acids or nucleic acid-biotin cross-linking.
U.S. Pat. Nos. 5,272,056 and 5,504,075 disclose a Ni(TMAPES) complex but fails to disclose attaching a detectable label to this complex. These two patents are incorporated herein by reference in their entirety.
As the literature described above shows, no salicylimine containing nickel complex is known in the prior art for the application of directly detecting or isolating nucleic acids. There is a need in the art for more effective method of studying chromosome structure, detecting specific nucleotide bases, and studying protein-DNA interactions.
The invention provides a salicylimine containing nickel complex for detecting protein-nucleic acid interactions and the presence of single-stranded regions of nucleic acids formed during gene expression, macromolecular assembly and chromatin reorganization.